The invention relates to the production of virosome-like-particles.
Vaccines against membrane-containing (enveloped) viruses mostly consist of killed or live attenuated viruses, or a preparation of their proteins (e.g. split virus vaccines or subunit preparations). Vaccination with killed viruses and protein preparations is safer than vaccination with live attenuated viruses, because the latter may mutate or revert back to wild-type virus. Subunit vaccines have the clear advantage that they can be prepared from viral proteins expressed by cells rather than from virus, making production safer and eliminating the risk of contaminating vaccine preparations with live viruses. However, while the injection of live viruses generally induces a strong immune response, protecting against future infections by the virus, protein preparations may fail to do so. This is because live attenuated viruses infect cells of the body, and will be replicated by these cells to some extent, after which the infected cells and viruses are detected by cells of the immune system, triggering an immune response. Live or killed viruses can also be taken up by specialized phagocytic cells of the immune system, such as dendritic cells, and be presented to other cells of the immune system, triggering an immune responses. These phagocytic cells patrol the body, ingesting particles of the size of viruses all the time, but they do not efficiently take up the purified proteins of split virus or subunit vaccines [1-2].
Numerous attempts to reinforce the immune response to subunit preparations by physical or chemical means have been undertaken. The most important principle that emerges from these experiments is that multiple copies of the viral proteins need to be combined in particles, that will be taken up efficiently by phagocytic cells. These particles can be virosome-like-particles, whole virosomes, Immune-Stimulating Complexes (ISCOMs), proteosome preparations or proteins on microparticle carriers. Frequently, these particles also contain chemical substances that are meant to stimulate the immune system (called adjuvants), which address specific receptors on the phagocytes or the effector cells of the immune system.
For example, ISCOMs are cage-like particles containing viral proteins complexed with adjuvants such as saponins like Quil A®), usually isolated from the bark of Quillaia sopanaria Molina. Mixed with antigen, and lipids such as cholesterol, these adjuvants form the typical ISCOM structures of between 30-40 nm, rendering the antigen sufficiently particulate for uptake by phagocytic cells of the immune system, while acting at the same time as an adjuvant. However, although ISCOMs have been used in a number of veterinary vaccines, and strongly enhance the immunogenicity of the viral membrane proteins, the development of such vaccines for humans has been inhibited by concerns about their toxicity and the complexity of the mixture [3]. A more recent type of particle, proteosomes (US application 0010053368) [4], consists of complexes of antigenic proteins such as the influenza hemagglutinin or the human immunodeficiency virus envelope glycoprotein, mixed with the purified outer membrane proteins of bacteria such as meningococci. While these multiple bacterial proteins may act as adjuvants, the complex nature of such mixtures, consisting of multiple proteins, lipids and other substances, will present a regulatory issue. Moreover, the immune response is directed against all proteins and other antigens present in the solution, and less specifically against the viral proteins.
A particularly useful kind of vaccine composition which has been developed in the art is known as ‘virosomes’, which are lipid bilayers containing viral glycoproteins derived from enveloped viruses. The concept of using such virosomes for vaccination purposes, particularly for vaccination against influenza, has been introduced by Almeida et al. [5]. Virosomes (or virosome-like-particles, considering that the exact size and shape of the particles is less important than that their particulate nature and functional and biologically-relevant membrane fusion activity is retained) are generally produced by extraction of membrane proteins and lipids from enveloped viruses with a detergent, followed by removal of this detergent from the extracted lipids and viral membrane proteins, in fact reconstituting or reforming the characteristic lipid bilayers (envelopes) that surround the viral core or nucleocapsid [5].
Influenza virus and Semliki Forest virus (SFV) are two classical examples of enveloped viruses. The first step in infection of cells by these viruses is uptake of intact viral particles by receptor-mediated endocytosis. Inside the endosomal compartment the conditions are mildly acidic due to the activity of a membranous ATP-dependent proton pump. Under these conditions (pH 5-6), the viral spike proteins undergo a conformational change which results in triggering of viral membrane fusion activity. Subsequent fusion of the viral membrane with that of the endosome results in cytoplasmic penetration of the viral genome, and the cell can be considered infected [6].
Enveloped viruses in general carry specific membrane proteins (the “spikes”) which are required for binding to and entry of cells. For example, influenza virus carries about 500 copies of hemagglutinin (HA), which is composed of two disulfide-linked subunits, HA1 and HA2, and which forms trimers in the viral membrane [7]. The HA1 subunits form the top domain of the spike and carry a pocket responsible for binding of the virus to its plasma membrane receptor, sialylated lipids (gangliosides) and proteins. The stem region of the spike is mainly composed of the three HA2 subunits. Each HA2 subunit contains a N-terminal fusion peptide, a highly conserved apolar sequence. Upon the conformational change induced by exposure to mildly acidic pH these peptides interact with the target membrane leading to fusion [6].
While both SFV and influenza enter cells through receptor-mediated endocytosis and fusion from within acidic endosomes, the molecular mechanisms of membrane fusion mediated by SFV and influenza virus are quite different. Each Semliki Forest virion contains 80 spikes, which are each composed of three E1/E2 heterodimers [8]. These two membrane proteins have separate functions during the viral life cycle. Thus, while E2 is involved in virus-receptor binding, E1 mediates the merging of the viral and endosomal membranes. After acidification the E1/E2 complex dissociates and E1 rearranges to form homotrimers, and while influenza HA has a well-defined N-terminal fusion peptide, E1 does not. Another prominent difference between both viruses is that HA-mediated fusion is not very sensitive to target membrane lipid composition [9]. Fusion of SFV has a strict requirement for the presence of cholesterol [11-12] and sphingolipid [13-17] in the target membrane.
An essential feature of virosome-like-particles obtained by reconstitution (herein also called virosomes) is that they are particles of the size that is efficiently taken up by phagocytic cells of the immune system, and they closely mimick the composition, surface architecture and functional activities of the native viral envelope. Virosomes that are particularly active in inducing an immune response were found to have maintained the proper functions of the envelope proteins of the native virus, such as membrane fusion, receptor-binding and other activities. Preservation of receptor-binding and membrane fusion activity is essential for expression of full immunogenic properties of said virosomes.
In the process used for the formation of virosomes, the viral membrane (envelope) is reformed during detergent removal. This step is thought to be necessary for a functional reconstitution of the native viral envelope, but quite difficult to control. Current detergent removal protocols that result in reconstitution are mostly based on detergents with a low critical micelle concentration (cmc), and such detergents are particularly difficult to remove, in contrast to detergents with a comparatively high cmc, which can be removed by dialysis or ultrafiltration. However, it was found that the latter type of detergent does not generally properly reconstitute viral membrane proteins, including the influenza virus hemagglutinin, leading predominantly to the formation of empty membranes on the one hand, and protein aggregates on the other.
Previously we have developed a method for the reconstitution of influenza virus HA [17,18]. This method is based on solubilisation of the virus membrane with the nonionic detergent octaethyleneglycol-n-dodecyl monoether (C12E8), and, after sedimentation of the viral nucleocapsid by ultracentrifugation, removal of the detergent from the supernatant by a hydrophobic resin (Bio-Beads SM-2). The vesicles formed in this manner have been identified as virosomes. The approach allows the introduction of reporter molecules in either the lipid bilayer or the aqueous interior of the virosomes. For this purpose, we have used a fluorescent lipid, pyrene-labeled phosphatidylcholine (pyrPC), incorporated in the virosome membrane during reconstitution, to quantitatively measure membrane fusion between virosomes and erythrocyte ghosts [18-20] or target liposomes [21]. In addition, we have encapsulated water-soluble reporter molecules, gelonin [22] and the A chain of Diphtheria toxin [18,23], in virosomes and delivered these substances to target cell cytosol. These studies, and later studies in WO 92/19267 have indicated that after C12E8-mediated reconstitution influenza virus HA has substantially retained its original activity. It is, however, well recognized that, although such virosomes can elicit strong, protective, immune responses (e.g. WO 88/08718 and WO 92/19267), alternative methods are required that allow the efficient production of functionally reconstituted viral envelopes on an industrial scale. However, removal of detergent from the supernatant by a hydrophobic resin can hardly be scaled up sufficiently.
Retention of biologically-relevant fusion activity represents the only rigorous criterion for the functional reconstitution of viral envelopes. So far, the use of detergents like C12E8 and Triton X-100, which have a low critical micelle concentration (cmc), appear to represent the method of choice for the functional reconstitution of viral envelopes [18]. However, the use of low-cmc detergents has a disadvantage in that they cannot be readily removed by dialysis. For this reason, many reconstitution procedures rely on the use of detergents with a relatively high cmc. A widely used detergent in this category is the nonionic n-octyl-β-D-glucopyranoside, or octylglucoside, which has a cmc of about 20-25 mM. In most hands, e.g. ours and in WO 92/19267, however, attempts to reconstitute HA from octylglucoside-solubilised virus were unsuccesful in a variety of conditions, and no substantially fusogenic particles were obtained [17, 18].